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Biomedical subjects

A Kuliev

Publications and source records attributed to A Kuliev.

At least 37 records · Page 2Linked to original sources
PubMed1999

Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes.

PURPOSE: A number of pitfalls in single-cell DNA analysis, including undetected DNA contamination, undetected allele drop out, and preferential amplification, may lead to misdiagnosis in preimplantation genetic diagnosis of single-gene disorders. METHODS: Preimplantation genetic diagnosis was performed by sequential first and second polar body analysis of oocytes in 26 couples at risk for having children with various single-gene disorders. Mutant genes were amplified simultaneously with linked polymorphic markers, and only embryos resulting from the mutation-free oocytes predicted by polar body analysis with confirmation by polymorphic marker testing were transferred back to patients. RESULTS: Overall 529 oocytes from 48 clinical cycles (26 patients) were tested, resulting in the transfer of 106 embryos in 44 clinical cycles. As many as 46 (9.6%) instances of allele dropout were observed, the majority (96%) of which were detected. Seventeen unaffected pregnancies were established, of which nine resulted in the birth of an unaffected child, and the rest are ongoing. CONCLUSIONS: A high accuracy of preimplantation genetic diagnosis of single-gene disorders is achieved by application of sequential analysis of the first and second polar body and multiplex polymerase chain reaction.

Alleles↗
PubMed1999

Birth of healthy children after preimplantation diagnosis of thalassemias.

BACKGROUND: Preimplantation genetic diagnosis (PGD) allows couples at risk of having children with thalassemia to ensure the healthy outcome of their pregnancy. METHODS: Seventeen PGD clinical cycles were initiated for Cypriot couples at risk of having children with different thalassemia mutations, including IVSI-110, IVSI-6, and IVS II-745. Unaffected embryos for transfer were selected by testing oocytes, using first and second polar body (PB) removal and nested polymerase chain reaction analysis followed by restriction digestion. RESULTS: Unaffected embryos were selected in 16 of 17 PGD cycles. Of 166 oocytes studied from these cycles, 110 were analyzed by sequential analysis of both the first and the second PB, resulting in preselection and transfer of 45 unaffected embryos. This resulted in seven pregnancies and in the birth of five healthy thalassemia-free children. The embryos predicted to have inherited the affected allele were not transferred. Analysis of these embryos confirmed the PB diagnosis. CONCLUSIONS: Sequential first and second PB testing of oocytes is reliable for PGD of thalassemia and is a feasible alternative to prenatal diagnosis in high-risk populations.

Alleles↗
PubMed1999

Construction and sequence analysis of subtraction complementary DNA libraries from human preimplantation embryos.

PURPOSE: Because stage-specific genetic expression in human preimplantation development is not sufficiently studied, we have undertaken the construction of a subtraction complementary DNA (cDNA) library enriched for transcripts specific for human blastocysts. METHODS: For this purpose individual pools of cDNAs synthesized from four hatched blastocysts and three cleaving 8- to 10-cell embryos were exposed to suppression subtractive hybridization to minimize the presence of transcripts of housekeeping genes and other genes of maternal origin known to be expressed earlier in preimplantation development. Random clones of this library were sequenced and analyzed using the BLAST algorithm. RESULTS: The resulting subtraction library had a complexity of 3 x 10(5) and an average size of inserts of about 0.8 kb. Sequencing of random library clones revealed the following human genes: CD9 antigen, fatty acid binding protein, ferritin heavy chain, amyloid precursor, MAP kinase messenger RNAs, DNA clone 127H14, messenger RNA for diacylglycerol kinase, a sequence homologous to C1 inhibitor, messenger RNA for the KIAA0145 gene, and others. CONCLUSIONS: The presence of these genes in human preimplantation development suggests expression specific to the blastocyst stage.

Algorithms↗
PubMed1999

Prepregnancy testing for single-gene disorders by polar body analysis.

Preventive measures for single-gene disorders are currently based on carrier screening in pregnancy and prenatal diagnosis. Although this has been extremely effective for preventing new cases of common inherited conditions, the major limitation is still termination of 25% of wanted pregnancies following detection of affected fetuses. To overcome this important problem, we developed a method for prepregnancy genetic testing that involves DNA analysis of the first and second polar bodies, which are extruded during maturation and fertilization of oocytes. We offered this option to 28 couples at risk for having children with single-gene disorders. Fifty clinical cycles were performed from these patients for the following conditions: 20 for cystic fibrosis, 18 for thalassemia, 6 for sickle cell disease, 2 each for Gaucher disease and LCHAD (long-chain 3-hydroxyacyl-COA dehydrogenase deficiency), and 1 each for hemophilia B and phenylketonuria. Oocytes obtained from these patients using in vitro fertilization procedures (IVF) were tested by a sequential multiplex nested PCR analysis of the first and second polar body to detect the gene involved simultaneously with linked polymorphic markers. A total of 191 of 399 oocytes with predicted genotype were mutation free and preselected for fertilization and transfer. In all but three cycles, one to three unaffected embryos with predicted unaffected genotypes were transferred, resulting in 20 pregnancies, from which 19 healthy children have been born. The follow-up analysis of embryos resulting from oocytes with predicted affected genotype, confirmed the diagnosis in 97% of cases, demonstrating the reliability of prepregnancy diagnosis of single-gene defects by polar body analysis.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1998

Three births after preimplantation genetic diagnosis for cystic fibrosis with sequential first and second polar body analysis.

OBJECTIVE: The purpose of this study was to determine the accuracy and feasibility of sequential polar body removal and analysis for preimplantation genetic diagnosis of mendelian disorders. STUDY DESIGN: Three couples with risk factors for cystic fibrosis had preimplantation genetic diagnosis with the use of sequential polar body analysis. After stimulation, oocytes were harvested and the first polar bodies were removed and analyzed on the day of aspiration. The following day, after fertilization, the second polar bodies were aspirated. Only embryos known to have inherited the normal maternal allele were transferred. RESULTS: All three couples had successful pregnancies resulting in the births of unaffected infants. CONCLUSIONS: Preimplantation diagnosis with the use of sequential polar body removal is feasible and can prevent the establishment of genetically abnormal pregnancies for couples at risk.

Alleles↗
PubMed1998

Sequence analysis of libraries from individual human blastocysts.

PURPOSE: It has recently become possible to construct cDNA libraries from individual human blastocysts to investigate the expression of embryonic genes in human preimplantation development. We have previously reported the expression of beta-actin, CD-59, and homeobox OCT-3 and identified almost-complete homology of sequences to human histone 3.1 and human ribosome protein S25. In the present paper, our further sequencing analysis of cDNA libraries from single human blastocysts is described. METHODS: cDNA libraries were constructed from 13 blastocysts. Sequence analysis was performed in 120 clones from one of these cDNA libraries with fragments of 50 to 1000 bp. Their sequence identity was analyzed using the expressed sequence tag (EST) database. RESULTS: The presence of two housekeeping genes, hexokinase I and serine/threonine phosphorylase, and four other ESTs was demonstrated, the identity of which, with particular gene expression in preimplantation development, has not yet been established. CONCLUSIONS: The data demonstrate the usefulness of constructing cDNA libraries from individual human blastocysts and their values in the analysis of genetic expression in human preimplantation development.

Blastocyst↗
PubMed1998

Allele dropout in polar bodies and blastomeres.

PURPOSE: Because allele dropout (ADO) is frequently observed in single-cell polymerase chain reaction analysis, it is important to develop a method for efficient detection of ADO, in order to avoid possible misdiagnosis in preimplantation diagnosis. METHODS: We introduced a simultaneous amplification of mutant genes and linked polymorphic markers, such as a 4-bp repeat (GATT) at the 3' end of intron 6 in the cystic fibrosis (CF) gene and a short tandem repeat at the 5' end of the beta-globin gene. Three types of single heterozygous cells were studied for the amplification of both alleles, including 150 blastomeres, 1615 fibroblasts, and 170 first polar bodies, obtained from patients at risk for having children with cystic fibrosis (delta F-508 mutation) or sickle cell disease. RESULTS: ADO rates of as high as 33.3% for delta F-508 mutation and 22.8% for beta-globin gene were observed in single blastomeres, compared to 7.1 and 7.7% in single fibroblasts and 5.9 and 9.6% in first polar bodies, respectively. The application of simultaneous amplification of the above linked polymorphic markers allowed detection of more than half of the cases of ADO in blastomeres (19.4% for cystic fibrosis and 12.3% for beta-globin gene) and almost all ADOs in polar bodies, particularly when the two-step sequential analysis of the first and second polar body was applied in preimplantation diagnosis of single gene disorders. CONCLUSIONS: Simultaneous amplification of linked polymorphic markers in single-cell DNA analysis of single-gene defects is an efficient method for avoiding the risk of misdiagnosis in preimplantation diagnosis.

Alleles↗
PubMed1998

Preimplantation diagnosis of thalassemias.

PURPOSE: Preimplantation genetic diagnosis (PGD) is an important option for couples at risk of having children with beta-globin mutations to avoid selective abortions of affected fetuses following prenatal diagnosis. METHODS: We performed PGD for thalassemia in 12 clinical cycles (IVS1-110, and IVS-745 mutations) using biopsy of the first and second polar bodies (PBs) extruded from oocytes during maturation and fertilization, coupled with nested polymerase chain reaction analysis and restriction digestion. RESULTS: A total of 118 oocytes was obtained, of which 78 had results for both the first and the second PBs. This resulted in the selection and transfer of 30 unaffected embryos (2.5 embryos per cycle). To avoid a possible misdiagnosis due to allele dropout (ADO), we have also introduced simultaneous detection of two highly polymorphic linked markers, a short tandem repeat immediately at the 5' end of the globin gene and HUMTH01 which is a syntenic short tandem repeat. The application of multiplex polymerase chain reaction of the beta-globin gene and linked polymorphic markers enabled detection of ADO in five first PBs, thus avoiding the transfer of potentially affected embryos resulting from their corresponding oocytes. CONCLUSIONS: Confirmation studies of the embryos resulting from the oocytes predicted to contain an affected gene confirmed the diagnosis in 98% of the cases, thus demonstrating the accuracy and reliability of PB PGD of thalassemia mutations. The application of PB analysis in six patients resulted in two ongoing pregnancies with a thalassemia-free fetus already confirmed in both of them by prenatal diagnosis.

Alleles↗
PubMed1998

Preimplantation diagnosis of common aneuploidies by the first- and second-polar body FISH analysis.

PURPOSE: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. METHODS: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. RESULTS: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15 pregnancies are ongoing after being confirmed normal by prenatal diagnosis. CONCLUSIONS: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.

Aneuploidy↗
PubMed1998

Preimplantation diagnosis of autosomal dominant retinitis pigmentosum using two simultaneous single cell assays for a point mutation in the rhodopsin gene.

A couple requested preimplantation genetic analysis for a dominant form of retinitis pigmentosum caused by a C-->A transversion in the rhodopsin gene. Since this point mutation does not alter a restriction endonuclease site we designed two separate analytical systems, one involving site-specific mutagenesis and the other involving allele-dependent length polymorphism. After establishing the accuracy and robustness of these assay systems we utilized both systems simultaneously in a heminested polymerase chain reaction (PCR) system. This allowed accurate preimplantation diagnosis to be performed. One embryo was transferred but a pregnancy did not occur.

Cells, Cultured↗
PubMed1998

Isolation of cDNA libraries from individual human preimplantation embryos.

Although available data from the mouse model suggest that morphogenesis during preimplantation development is dependent on the expression of embryonic genes, convincing data for human preimplantation embryos are missing. To investigate the expression of genes involved in human preimplantation development we constructed cDNA libraries from human individual blastocysts and screened them for the expression of beta-actin, CD59, homeoboxes OCT-3 and HOXA4, and HLA-G and hMLH-1 genes. Beta-actin, CD59, and OCT-3 were detected by reverse transcription-polymerase chain reaction (RT-PCR), while HOXA4, HLA-G and hMLH-1 were undetected. Sequencing of 48 random clones from two libraries revealed a different identity to the known genes including 99% identity to human histone 3.1 and human ribosome protein S25 complete cDNA. These data demonstrate the feasibility of constructing cDNA libraries from individual human preimplantation embryos and their potential usefulness in the assessment of the relevance of specific gene expression in the failures of preimplantation development.

Blastocyst↗
PubMed1998

The history of community genetics: the contribution of the haemoglobin disorders.

The emerging possibilities of applying new diagnostic technologies for genetic diagnosis and screening point to the need for a discipline of community genetics. Genetic population screening for haemoglobin disorders (thalassaemias and sickle cell disorders) has been practised on a large scale for over 20 years, and basic concepts and methods of community genetics have been developed within this framework, under the auspices of the WHO. Genetic population screening is a public health activity. It involves many disciplines, and planning depends on reliable and regularly updated information. Basic methods for describing genetic epidemiology in terms of service needs, and for regular service audit have been developed, and IT-based approaches for making simple, accurate and diagnosis-specific information accessible to the public and health professionals are being explored.

Journal Article↗
PubMed1998

Detection of translocations involving the Y-chromosome in prospective prenatal screening of common chromosomal aneuploidies by FISH.

In the application of the fluorescence in situ hybridization (FISH) technique for prospective prenatal screening of common aneuploidies involving the autosomes 13, 18, and 21, and sex chromosomes, six cases of inconsistency between the results of FISH analysis and the results of karyotyping of cultured amniocytes have been observed, including two cases of translocation involving the Y-chromosome and chromosome 15 in a total of 904 cases of amniocentesis studied. In one case, the translocation was of maternal origin, and in the other, of paternal origin. In both cases, the couples decided to continue the pregnancy and normal babies were delivered. The data show the usefulness of applying the FISH technique in prospective prenatal screening of common trisomies for the possible detection of rare chromosome rearrangements involving the Y-chromosome.

Aneuploidy↗
PubMed1997

Preimplantation diagnosis of single gene disorders by two-step oocyte genetic analysis using first and second polar body.

Previous work on preimplantation genetic diagnosis (PGD) of single gene disorders by the first polar body (IPB) analysis has demonstrated that the genotype of a considerable number of embryos resulting from heterozygous oocytes cannot be predicted without testing their second PB (IIPB). To overcome this limitation we introduce a two-step DNA analysis of oocytes using both IPB and IIPB to identify hemizygous mutation-free oocytes following the second meiotic division. In the application of the approach to PGD of cystic fibrosis (CF) Delta F-508 mutation, sickle cell disease, and hemophilia B, 80 oocytes were studied by both PBs, resulting in the identification and transfer of 32 homozygous normal embryos. A follow-up genotyping of 52 embryos, resulting from oocytes tested by both IPB and IIPB demonstrated the accuracy of the predicted genotypes. In addition to a nested PCR analysis of the mutant genes in PBs and resulting embryos, simultaneous amplification of different polymorphic markers was performed, demonstrating the reliability of the two-step polar body analysis of oocytes.

Alleles↗
PubMed1996

Preimplantation polar body diagnosis.

Preimplantation polar body diagnosis makes it possible to detect and avoid genetic and chromosomal disorders before pregnancy. We have shown that the polar body biopsy does not affect fertilization and viability of the resulting embryos. Our present experience of polar body diagnosis includes 187 clinical cycles, performed for preimplantation diagnosis of cystic fibrosis, alpha-1-antitrypsin deficiency, Tay-Sach's disease, retinitis pigmentosa, hemophilia A, Alport and sickle cell disease, and common aneuploidy, using the FISH technique. Over three-quarters of these cycles have resulted in embryo transfer, 38 in clinical pregnancy and 12 in the birth of an unaffected child. The present review describes the results of our clinical trial on the polar body diagnosis of genetic and chromosomal disorders, demonstrating the reliability of polar body genetic analysis for preimplantation diagnosis.

Anemia, Sickle Cell↗